Comparison of vascular growth factors in the murine brain reveals placenta growth factor as prime candidate for CNS revascularization by Emília Ilona Gaál, Tuomas Tammela, Andrey Anisimov, Serge Marbacher, Petri Honkanen, Georgia Zarkada, Veli-Matti Leppänen, Turgut Tatlisumak, Juha Hernesniemi, Mika Niemelä, and Kari Alitalo Blood Volume 122(5):658-665 August 1, 2013 ©2013 by American Society of Hematology

Effects of human vascular growth factor gene transduction on the brain vasculature. Effects of human vascular growth factor gene transduction on the brain vasculature. Immunostaining of the targeted brain regions 2 weeks after transduction. Quantification of (A) podocalyxin-positive areas. ***P < .001, when compared with HSA control; error bars show SD. (B-G) Examples of podocalyxin staining of (B) VEGF, (C) VEGF-B, (D) VEGF-C, (E) PlGF, (F) cAng1, and (G) HSA transduced brains. (B,D) Arrowheads point to hemangiomas. (C,E-G) Arrows point out needle path. Scale bar, 200 μm. Emília Ilona Gaál et al. Blood 2013;122:658-665 ©2013 by American Society of Hematology

Inflammatory response after vascular growth factor expression. Inflammatory response after vascular growth factor expression. (A) Quantitative analysis of inflammation by CD45-positive cell counting per injection site. ***P < .001, when compared with HSA control; error bars show SD. (B-G) Immunostaining for CD45-positive cells (red), vascular counterstaining for podocalyxin (green). Scale bar, 200 μm. Emília Ilona Gaál et al. Blood 2013;122:658-665 ©2013 by American Society of Hematology

Immunohistochemical evidence for mural cell recruitment after PlGF treatment. Immunohistochemical evidence for mural cell recruitment after PlGF treatment. Immunostaining of endothelial cells for (A,C,E,G,I,) podocalyxin (green) and for (B,D,F,H,J) pericytes (PDGFR-β, red). (K-L) Quantitative analysis of (K) PDGFR-β–positive area, and quantitative comparison of (L) podocalyxin vs PDGFR-β–positive areas, with horizontal line at 1, referring to equal area with both stainings. (E-H) Arrowheads point to hemangiomas. ***P < .001 when compared with HSA control; error bars show SD. Scale bar, 50 μm. Emília Ilona Gaál et al. Blood 2013;122:658-665 ©2013 by American Society of Hematology

Electron microscopic evidence for mural cell recruitment after PlGF treatment. Electron microscopic evidence for mural cell recruitment after PlGF treatment. (A-B,D) Transmission electron microscopic images of vessels newly formed upon PlGF gene transfer. (A-B) Microvessels are surrounded by 2 layers of (endothelial and pericyte) basal laminae (arrowheads). (B) Capillaries are in intimate contact with the astrocytes (As); arrow points to tight junction between 2 endothelial cells. (C) SMA-positive vessels (arrow) after hPlGF gene transfer, for quantification. (D) Quantification of SMA-positive vessel density from immunostainings. *P < .05; **P < .01; and ***P < .001 when compared with HSA control; error bars show SD. (E) Arterioles are ensheathed in a single layer of VSMC. EC, endothelial cell; Ne, neuron; Pe, pericyte; RBC, red blood cell; VSMC, vascular smooth muscle cell. Scale bars: A-B,E, 1 μm; C, 200 μm. Emília Ilona Gaál et al. Blood 2013;122:658-665 ©2013 by American Society of Hematology

Evaluation of lectin extravasation with optical projection tomography. Evaluation of lectin extravasation with optical projection tomography. Right-sided growth factor injections were performed accompanied by left-sided sham operation. After VEGF gene transfer, major extravasation can be observed at the site of the hemangioma (arrowhead: A,C,E). PlGF gene transfer induced the formation of a dense microcapillary network, without significant extravasation (arrow: B,D,F). Autofluorescence is shown in green; lectin staining of blood vessels and extravasation in blue. ca, caudal; Ro, rostral. Emília Ilona Gaál et al. Blood 2013;122:658-665 ©2013 by American Society of Hematology

Effects of vascular growth factor treatment on the glial and neuronal structures. Effects of vascular growth factor treatment on the glial and neuronal structures. (A-E) Qualitative analysis of astrocytes by immunostaining for GFAP. Scale bar, 200 μm. (F-J) Qualitative analysis of neuronal structures by immunostaining for neurofilaments. Nuclear staining with DAPI. Scale bar, 50 μm. Arrows point out needle path (A-B,E). Arrowheads point to hemangiomas and its side effects in the (C-D) glial and (H-I) neuronal structures. Emília Ilona Gaál et al. Blood 2013;122:658-665 ©2013 by American Society of Hematology

In vivo effects of VEGF and PlGF visualized by MRI In vivo effects of VEGF and PlGF visualized by MRI. Right-sided growth factor injections were performed accompanied by left-sided sham operation. In vivo effects of VEGF and PlGF visualized by MRI. Right-sided growth factor injections were performed accompanied by left-sided sham operation. Incubation time: 2 weeks. (A) After VEGF gene transfer, the macroanatomy of the brain is distorted as observed in T1 images, (C) the BBB is disintegrated from a large area seen as enhancement (white) on T1-Gd series, (E, continuous line: anatomical midline, dashed line: actual midline, black arrow: brain shift) T2 sequence shows major edema (white) with brain shift, and T2* demonstrates hemorrhage (black). Arrowheads point to a hemangioma. Note that after PlGF treatment, (B) the macroanatomy is intact, (D) enhancement is minimal, (F, continuous line: anatomical and actual midline coincide) there is no edema and no brain shift, and (H) minor hemorrhage is only seen at injection path. White arrows point to injection path. Emília Ilona Gaál et al. Blood 2013;122:658-665 ©2013 by American Society of Hematology